Proteolytic activities during growth and aging in the fungus Podospora anserina: Effect of specific mutations

In Podospora anserina five proteolytic enzymes were characterized by chromatographic procedures. Three of these (proteases A, B and C) were found in the cell extracts of growing cultures and the other two (proteases III and IV) were revealed by studies on protoplasmic incompatibility. During growth, only protease C, an acidic enzyme, was active in crude extracts. From the stationary and the poststationary stages this activity decreased and finally disappeared, whereas a neutral serine protease (activity B) became active in crude extracts. A close relationship was observed between the proteolytic activity of the culture filtrates and the intracellular protease(s) concomitantly active in the crude extracts. None of the proteases associated with protoplasmic incompatibility was detected, both in the extra- and intracellular spaces. Qualitative variations in the proteolytic activities during stationary and post-stationary stages depended on the presence of specific genes and mutations: the mod C mutation suppressing protoplasmic incompatibility, inhibits the progressive decrease of protease C and, furthermore, the presence of non allelic incompatibility genes have for consequence the substitution of serine protease B by serine protease A during the poststationary stage.     

The molecular mechanism of protoplasmic incompatibility and its relationship to the formation of protoperithecia in Podospora anserina.

In Podospora anserina, protoplasmic incompatibility due to interactions between non-allelic genes was suppressed by the effect of mutations in two modifier genes, mod-I and mod-2. It is shown that mod-I and mod-2 are involved in the production of three specific proteins, a phenoloxidase and two previously identified proteases (Bégueret & Bernet 1973 a) which are associated with the phenomenon of protoplasmic disintegration. These enzymes, whose messengers are normallly latent during vegetative growth, appear at this stage of the life cycle only as a consequence of incompatible gene interactions. The mode-I and mod-2 genes and each of the five incompatibility loci involved in non-allelic incompatibility systems also participate in the formation of the protoperithecia. This pleiotropic effect suggests that protoplasmic incompatibility is a deviation in the normal physiological processes of protoperithecial formation.     

Podospora anserina mutants defective in cell regeneration and ascospore germination: The presence of a possible lesion of the plasma membrane

In Podospora anserina three allelic mutations (of modD) were defective in ascospore germination, protoperithecium formation, and growth renewal from stationary cells. To investigate the action of the modD gene a mutation (modE) was selected, which suppresses the developmental defects associated with a modD mutation (modD(2)). The comparison of modD, modE, modD, modE, and wild-type strains gave the following results: (i) each mutant showed a specific and large spectrum of variations in the level of the in vivo resistance to unrelated drugs (cycloheximide, sorbose, thiourea, ethionine); (ii) the protoplasts isolated from the modE strain were osmotic fragile at elevated temperatures; (iii) the temperature at which the osmotic fragility associated with modE inhibits growth, in a medium of a standard osmotic pressure, was strongly dependent on modD mutations. Furthermore, the coupling of the modD(1) mutation to modC(1), a mutation which is known to inhibit growth at acid pH (6.5), resulted in a strain with a wild-type pH dependence. Considered together these results suggest that the modD developmental mutations could be, like modE, responsible for a modification of plasma membrane properties.     

Aerial organs and cell death inPodospora anserina mutants: Relationship with protoplasmic incompatibility

Mutations at six loci (lprA,B, C, D, E,andF) led to an increased size of mycelial aerial organs (protoperithecia and aerial hyphae) which was accompanied by a reduction in the life span of stationary phase cells. Despite having a common phenotype, the mutations acted either in protoperithecia and aerial hyphae (lprD) or in vegetative cells (lprA,B, C, E,andF). It is deduced thatlpr mutants in stationary phase die prematurely due to the uncontrolled expression of a function that converts vegetative cells into a source of nutrients to be translocated to the aerial organs. Thelpr mutant phenotype was suppressed by mutations inhibiting protoplasmic incompatibility and no recombination occurred betweenlprB and one incompatibility locus. These results suggest that protoplasmic incompatibility is a deviant expression of the cell death associated with the development of protoperithecia.     

A constitutive mutation in a posttranscriptional regulator gene for a phenoloxidase and proteases in Podospora anserina

A recessive mutation in the gene mod-2 results in the synthesis at low temperatures of a phenoloxidase and an arrest of growth, reversible by beta-phenyl-pyruvic acid, a protease inhibitor. Phenoloxidase synthesis is 5-fluorouracil resistant and cycloheximade sensitive. Suppression of both cold sensitivity and phenoloxidase synthesis by common factors (higher NH4+ concentrations or mutations) suggests that the protease, suspected to be responsible for cold sensitivity, also arises from preexisting mRNA molecules. Instead of being recessive and constitutive, the mod-2 mutations is suppressive and dominant when cold sensitivity and phenoloxidases synthesis are induced as the consequence of the nonallelic gene interactions C/D, C/E, or R/V responsible for protoplasmic incompatibility. Combinations of nonallelic incompatibility systems and several mod-2 mutations lead us to hypothesize that the translational control of the above proteins depends on conformational relationships between incompatibility gene products and mod-2.     

Protoplasmic Incompatibility in PODOSPORA ANSERINA: a Possible Function for Incompatibility Genes

The suppression of protoplasmic incompatibility resulting from nonallelic gene interactions has been obtained by the coupled effect of mutations in the modA and modB genes (Bernet 1971). Due to their female sterility, modA modB strains provide an experimental tool to determine whether or not the mod and incompatibility loci are involved in a function other than protoplasmic incompatibility. Present results show that modA modB female sterility is a nonautonomous trait since heterokaryotic mycelia that include a modA modB nucleus and a female fertile nucleus (wild-type, modA or modB) produce modA modB protoperithecia, which are also formed by culture on medium supplemented with specific amino acids. Using modA modB strains, which are sterile at 32 degrees and fertile at 26 degrees , we have shown that the mod genes have no specific sequential timing. Indeed, the mod mutations may prevent the achievement of the female sexual cycle at any developmental stage from before early differentiation of protoperithecia until ascospore maturation. Employing different modA and modB mutations, we have shown that protoperithecia in modA modB cultures are generally distributed in female fertile rings; this result indicates that protoperithecia occur only in mycelial areas that have a restricted range of age at the time that modA modB thalli complete growth. Furthermore, nonsense mutations of incompatibility genes suppress the modA modB female fertile rings or restrict their width, suggesting that incompatibility loci, like the mod loci, are involved in protoperithecium formation. Taken together, these results lead to the postulate that mod and incompatibility genes do not determine, sensu stricto, protoperithecial function, as previously supposed (Boucherie and Bernet 1974), but may be involved in the homeostatic control of stationary cell functions essential for the complete development of the female sexual cycle.     

Use of human perivascular stem cells for bone regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized). FSDs are bicortical and are stabilized with a polyethylene bar and K-wires. The FSD described is also a critical size defect, which does not significantly heal on its own. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.     

A New Method for the Screening of Mutations Related to Protoplasmic Incompatibility, Differentiation and Plasma Membrane in the Fungus PODOSPORA ANSERINA

In Podospora anserina previous investigations showed that mutations in genes involved in the control of protoplasmic incompatibility cause defects at various stages of differentiation during the life cycle and also modify properties of the plasma membrane. To establish these relationships in another way, a new method for screening mutations has been developed as a first step. Eighty-five new mutants were selected for resistance to toxic products (sorbose or thiourea); in a second step these mutants were tested for modifications of protoplasmic incompatibility and cellular differentiation. Seven of the sorbose or thiourea resistant-mutants (i.e., 8%) exhibited new patterns of protoplasmic incompatibility. Genetic analyses were carried out with three mutants. Mutation X25 suppresses protoplasmic incompatibility resulting from all allelic interactions and restores the fertility of the crosses ♀ V x ♂ V1 and ♀ Z1 x ♂ Z2. Mutation V41 is an allele of the v locus with new properties. Mutation X61 totally suppresses the V/V'1 interaction and weakens all of the other allelic incompatibility systems; X61 strains are defective in protoperithecia differentiation. Electrophoresis of plasma membrane proteins showed that X61 strains lack two polypeptides whose apparent molecular weights are 41,000 and 44,000. This new screening method is especially efficient for obtaining new mutants and identifying additional genes involved in incompatibility. These results provide further support demonstrating the relationships between protoplasmic incompatibility, cellular differentiation and plasma membrane.     

Cell Physiological Studies on the Avena Coleoptile Treated with Ribonuclease

It was revealed with excised Avena coleoptile that the growth promoting effect of indole-3-acetic acid was inhibited by pretreatment with ribonuclease (Masuda 1959a, b). This effect of ribonuclease was presumed to involve its digestive action on the ribonucleic acid at the protoplasmic surface (Masuda 1959b). Ribonuclease treatment decreases the cation binding capacity of the ribonucleic acid at the protoplasmic surface (Masuda 1959a).
On the other hand, it has been confirmed that indole-3-acetic acid bas a remarkable effect on the physico-chemical properties of protoplasmic surface such as permeability (Masuda 1955) and adhesiveness of protoplasm to the cell wall (Masuda 1957, Masuda and Takada 1957).
The purpose of the present study is to see the effect of ribonuclease on some protoplasmic properties of cells of Avena coleoptile and substantiate the authors view on the participation of ribonucleic acid in the cell elongation.     

Phosphatidate phosphatase activity plays key role in protection against fatty acid-induced toxicity in yeast

The PAH1-encoded phosphatidate (PA) phosphatase in Saccharomyces cerevisiae is a pivotal enzyme that produces diacylglycerol for the synthesis of triacylglycerol (TAG) and simultaneously controls the level of PA used for phospholipid synthesis. Quantitative lipid analysis showed that the pah1Δ mutation caused a reduction in TAG mass and an elevation in the mass of phospholipids and free fatty acids, changes that were more pronounced in the stationary phase. The levels of unsaturated fatty acids in the pah1Δ mutant were unaltered, although the ratio of palmitoleic acid to oleic acid was increased with a similar change in the fatty acid composition of phospholipids. The pah1Δ mutant exhibited classic hallmarks of apoptosis in stationary phase and a marked reduction in the quantity of cytoplasmic lipid droplets. Cells lacking PA phosphatase were sensitive to exogenous fatty acids in the order of toxicity palmitoleic acid > oleic acid > palmitic acid. In contrast, the growth of wild type cells was not inhibited by fatty acid supplementation. In addition, wild type cells supplemented with palmitoleic acid exhibited an induction in PA phosphatase activity and an increase in TAG synthesis. Deletion of the DGK1-encoded diacylglycerol kinase, which counteracts PA phosphatase in controlling PA content, suppressed the defect in lipid droplet formation in the pah1Δ mutant. However, the sensitivity of the pah1Δ mutant to palmitoleic acid was not rescued by the dgk1Δ mutation. Overall, these findings indicate a key role of PA phosphatase in TAG synthesis for protection against fatty acid-induced toxicity.     

Transient neonatal denervation alters the proliferative capacity of myosatellite cells in dystrophic (129ReJdy/dy) muscle.

It has been previously shown that transiently denervated, neonatal dystrophic muscle fails to undergo the degeneration-regeneration cycle characteristic of murine dystrophy (Moschella and Ontell, 1987). Thus, the myosatellite cells (myogenic stem cells) in these muscles have been spared the mitotic challenge to which dystrophic myosatellite cells are normally subjected early in the time course of the disease. By in vitro evaluation of the proliferative capacity of myosatellite cells derived from extensor digitorum longus (EDL) muscles of 100-day-old genetically normal (+/+) and genetically dystrophic [dy/dy (129ReJdy/dy)] mice and from muscles of age-matched mice that had been neonatally denervated (by sciaticotomy) and allowed to reinnervate, it has been possible to directly determine whether the cessation of spontaneous regeneration in older dy/dy muscles in vivo, is due to an innate defect in the proliferative capacity of the myosatellite cells or exhaustion of the myosatellite cells' mitotic activity during the regenerative phase of the disease. This study demonstrates that transient neonatal denervation of dystrophic muscle (Den.dy/dy) increases the number of muscle colony-forming cells (MCFs) per milligram of wet weight muscle tissue, increases the plating efficiency, and significantly increases the in vitro mitotic activity of dystrophic myosatellite cells toward normal values. The increased mitotic capability of myosatellite cells derived from Den.dy/dy muscle as compared to unoperated dy/dy muscle suggests that there is no innate defect in the proliferative capacity of the myosatellite cells of dy/dy muscles and that the cessation of spontaneous regeneration in the dy/dy muscles is related to the exhaustion of their myosatellite cells' mitotic capability.     

Reverse reaction of malic enzyme for HCO3- fixation into pyruvic acid to synthesize L-malic acid with enzymatic coenzyme regeneration

Malic enzyme [L-malate: NAD(P)(+) oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)(+)). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO(3)(-) fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD(+), and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO(3)(-) and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30 degrees C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38 mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD(+).     

Bloom''s syndrome cells have an abnormal serum growth response

Certain growth responses of Bloom's syndrome (BS) dermal fibroblasts have been compared to those of normal human fibroblasts. By applying the principles of Michaelis-Menton kinetics to clonal dose-response data, serum and epidermal growth factor (EGF) requirements of the two cell types were found to be similar. However, the maximal clonal growth rate of BS cells was significantly lower than that of their normal counterparts. Although specific EGF binding by BS cells was marginally higher than in normal cells, EGF's growth-promoting activity was only half of that seen in normal cells. These observations indicate that the abnormally low growth rate of BS cells is not attributable to excessive requirements for serum-derived growth factors and suggest instead that the genetic defect in some way impairs the cells' ability to respond fully to growth stimulation.     

Dynamics of microbial propagation: Models considering inhibitors and variable cell composition

Mathematical models for microbial growth in batch and continuous cultures are formulated. The models have been referred to as distributed models since the microbial population in a culture is looked upon as protoplasmic mass distributed uniformly throughout the culture. Growth is regarded as the increase in this mass by conversion of medium components into biological mass and metabolic products. Two sets of models have been presented. The first arise from introducing additional considerations into the model proposed by Monod to account for the stationary phase and the phase of decline in a batch culture. These have been referred to as unstructured, distributed models since they do not recognize any form of structure in the protoplasmic mass. The models in the second set are referred to as structured, distributed models. Structure is introduced by considering the protoplasmic mass to be composed of two groups of substances which interact with each other and with substances in the environment to produce growth. The structured models account for the dependence of growth on the past, history of the cells; thus they predict all growth phases observed in batch cultures, whereas the unstructured models do not predict a lag phase. The full implications of the models for continuous propagation, as determined by the method of stability analysis and transient calculations, are discussed. The models prediet a number of new results and should be confronted with experiments.     

Accelerated hematopoietic stem cell aging in a mouse model of dyskeratosis congenita responds to antioxidant treatment

Mutations in DKC1, encoding telomerase associated protein dyskerin, cause X-linked dyskeratosis congenita (DC), a bone marrow (BM) failure, and cancer susceptibility syndrome. Decreased accumulation of telomerase RNA resulting in excessive telomere shortening and premature cellular senescence is thought to be the primary cause of disease in X-linked DC. Affected tissues are those that require constant renewal by stem cell activity. We previously showed that in Dkc1(Δ15) mice, which contain a mutation that is a copy of a human mutation causing DC, mutant cells have a telomerase-dependent proliferative defect and increased accumulation of DNA damage in the first generation before the telomeres are short. We now demonstrate the presence of the growth defect in Dkc1(Δ15) mouse embryonic fibroblasts in vitro and show that accumulation of DNA damage and levels of reactive oxygen species increase with increasing population doublings. Treatment with the antioxidant, N-acetyl cysteine (NAC), partially rescued the growth disadvantage of mutant cells in vitro and in vivo. Competitive BM repopulation experiments showed that the Dkc1(Δ15) mutation is associated with a functional stem cell defect that becomes more severe with increasing age, consistent with accelerated senescence, a hallmark of DC hematopoiesis. This stem cell phenotype was partially corrected by NAC treatment. These results suggest that a pathogenic Dkc1 mutation accelerates stem cell aging, that increased oxidative stress might play a role in the pathogenesis of X-linked DC, and that some manifestations of DC may be prevented or delayed by antioxidant treatment.     

Detection of an Intracellular Protease Inhibitor in Archaea

Proteolytic activity and a subtilisin inhibitor (NSI) were detected in Natrialba magadii cells. The proteolytic activity was due to two different proteases: a ∼90-kDa metallo protease (NMP) produced during exponential growth and a 246-kDa serine protease (NSP) detected in the stationary phase. Both proteases were detected in the cytosolic fraction. NSI activity was maximal during early stages of growth and decreased in the stationary phase. NSI is a 35-kDa thermosensitive protein; it inhibits NSP activity but has no effect on NMP, and it was detected as a soluble or membrane-bound protein depending on the growth phase. Our results suggest that NSI may regulate NSP activity in vivo and that this protease may have a role in stationary phase cells. To our knowledge, this is the first report on the occurrence of protease inhibitors in Archaea. Received: 4 May 2002 / Accepted: 10 July 2002